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J Biol Chem, Vol. 273, Issue 26, 16265-16272, June 26, 1998

Sites for G Binding on the G Protein  Subunit Overlap with Sites for Regulation of Phospholipase C and Adenylyl Cyclase

Ying Li, Pamela M. Sternweis^§, Sara Charnecki, Temple F. Smith^¶, Alfred G. Gilman^§, Eva J. Neer, and Tohru Kozasa^§

From the  Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115, the ^¶ Biomolecular Engineering Research Center, Boston University, Boston, Massachusetts 02111, and the ^§ Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235

Heterotrimeric G proteins, composed of  and subunits, forward signals from transmembrane receptors to intracellular effector enzymes and ion channels. Free activates downstream targets, but its action is terminated by association with GDP-liganded  subunits. Because  can inhibit activation of many effectors by , it is likely that the  subunit binding surfaces on overlap the surfaces necessary for effector activation. To test this hypothesis, we mutated residues on  shown to contact  in the recently published crystal structures of the heterotrimer (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319.). The  subunit binds to the flat, top surface of the toroidal  subunit and also extends a helix along the side of the  subunit at blade 1. We mutated four residues on the top surface of  (H₁[L117A], H₁[D228R], H₁[D246S], and H₁[W332A]) and two residues on the side of that contacts  (H₁[N88A/K89A]). Each of the mutant proteins was able to form dimers, but they differed in their ability to bind  and to activate phospholipase C ₂ (PLC₂), PLC₃, and adenylyl cyclase II. Mutation of residues along the side of the torus at blade 1 diminish affinity for  but do not prevent activation of any of the effectors. Mutations on the  binding surface differentially affected PLC₂, PLC₃, and adenylyl cyclase II. Residues that affect PLC and adenylyl cyclase II activity are found on opposite sides of the central tunnel, suggesting that PLC and adenylyl cyclase, like the  subunit, make many contacts on the top surface. None of the mutations affected the ability of to inhibit adenylyl cyclase I. We conclude that , PLC₂, PLC₃, and adenylyl cyclase II share an interaction on the top surface of . The importance of individual residues is different for  binding and for effector activation and differs even between closely related isoforms of the same effector.

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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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